JP2018526018A - Non-invasive method for assessing gene integrity of pluripotent stem cells - Google Patents

Non-invasive method for assessing gene integrity of pluripotent stem cells Download PDF

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JP2018526018A
JP2018526018A JP2018512901A JP2018512901A JP2018526018A JP 2018526018 A JP2018526018 A JP 2018526018A JP 2018512901 A JP2018512901 A JP 2018512901A JP 2018512901 A JP2018512901 A JP 2018512901A JP 2018526018 A JP2018526018 A JP 2018526018A
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ヴォス,ジャン デ
ヴォス,ジャン デ
アス,セド
ジロー,ニコラ
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インセルム(インスティチュート ナショナル デ ラ サンテ エ デ ラ リシェルシェ メディカル)
インセルム(インスティチュート ナショナル デ ラ サンテ エ デ ラ リシェルシェ メディカル)
ユニヴェルシテ ド モンペリエ
ユニヴェルシテ ド モンペリエ
センター ホスピタリエ ユニバーシタイア ド モンペリエ
センター ホスピタリエ ユニバーシタイア ド モンペリエ
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Abstract

The present invention relates to a novel non-invasive method for assessing the quality of cultured pluripotent stem cells. More specifically, the present invention is a non-invasive method for assessing the genetic integrity (eg, the presence of CNV) of cultured pluripotent stem cells by assessing cell-free nucleic acids in the supernatant of cell cultures. Regarding the method. [Selection] Figure 1

Description

  The present invention relates generally to the field of regenerative medicine. More specifically, the present invention relates to non-invasive methods and kits for determining the quality of pluripotent stem cells. More specifically, the present invention relates to non-invasive methods and kits for assessing the gene integrity of cultured pluripotent stem cells.

  Research on human pluripotent stem cells (hPSCs) is to assist in understanding and treating diseases affecting various cell types in the body by creating human cells for transplantation and drug exploration Providing a new tool to enable PSC (isolated from the inner cell mass of discarded embryos, ie human embryonic stem cells (hESC), or derived from differentiated cells, ie artificial ( Induced pluripotent stem cells (iPSCs)) have an amazing ability to proliferate. At a practical level, this means that enough cells can be made from one cell line to prepare thousands or even hundreds of thousands of therapeutic cells. Several clinical trials using differentiation-inducing cells of hESC have been or are currently being conducted: Geron Corporation (NCT01217008) has tested the safety of hESC-derived oligodendrocytes in patients with spinal cord injury. Advanced Cell Technology (ACT) has shown the safety of hESC-derived retinal pigment epithelial (RPE) cell therapy, Stargardt's Macular Dystrophy (SMD) (USA Trial: NCT01345002; UK Trial: NCT9832 The Korean clinical trial: NCT01625559), and the dry age-related Macular Degeneration (USA clinical trial: NCT01344993; Korean clinical trial: NCT01674829). Viacyte is testing the safety and efficacy of insulin-producing cells in subjects with type I diabetes (USA trial: NCT02239354). Philip Menasche has started a trial of transplanting progenitor cells derived from human embryonic stem cells in severe heart failure (NCT02057900). Pfizer (NCT01691261) is investigating the safety of using transplanted retinal cells derived from hESC to treat patients with advanced Stargardt disease. Finally, a study recently started in Japan by Masayo Takahashi of RIKEN, is derived from self-induced pluripotent stem cells (iPSCs) in patients with wet (wet) age-related macular degeneration (AMD) The safety of transplantation of retinal pigment epithelium (RPE) cell sheets is being investigated.

  All these clinical trials reveal that the biomedical potential is enormous, but there are still some practical problems to be solved. One of the biggest concerns is genetic abnormality.

  Genetic abnormalities are a major concern for using hPSC in regenerative medicine. If hPSC clones show genetic abnormalities, these cells and their differentiated progeny may not be able to faithfully reproduce normal adult tissue physiology and may pose a threat to the use of these cells in clinical settings There is even. Thus, it is essential to determine the cause and extent of genetic abnormalities in such cells. Genetic aberrations can be divided into two categories: those induced by cell culture and those induced by the cell reprogramming process.

  Human ESCs are karyotypically normal at the time of induction; however, aneuploid hESC clones may appear in cell culture. Since 2004, several studies have reported that the culture conditions used to amplify undifferentiated hPSCs have a significant effect on chromosome stability. Such chromosomal abnormalities are often recurrent. Increases in chromosome 12 (most often 12p), 17 (especially 17q), 20 or X were often detected using standard cytogenetic methods (G-banding) (Draper et al. al., 2004). Extensive testing of 40 hESC lines analyzing 1163 karyotypes concluded that 12.9% of hESC cultures showed chromosomal abnormalities (Taapken et al., 2011). Over the past five years, array-based technology (also called virtual karyotype) has improved the resolution of detection of genomic changes. Array-based comparative genomic hybridization (aCGH) or single nucleotide polymorphism (SNP) -arrays enable the identification of small-sized genomic abnormalities and the frequency of DNA changes in hPSCs It has even become clear that it may be much higher than previously thought (Laurent et al., 2011; Narva et al., 2010). Of these small chromosomal changes, a recurrent copy number variant (CNV) at the position of chromosome 20q11.21 has been identified (Lefort et al., 2008; Spits et al., 2008). ). This 20q11.21 region is also amplified in various cancers. Furthermore, it has been shown that the acquisition of 20q11.2 occurs early in cervical cancer. Point mutations are also involved in the adaptation process. More recently, whole-exome or whole-genome re-sequencing provided an unprecedented solution for identifying single base pair mutations in hPSC (Cheng et al., 2012 Funk et al., 2012; Gore et al., 2011). The generation of iPS by cell reprogramming leads to other potential sources of mutation. CGH microarray (Martins-Taylor and Xu, 2010; Pasi et al., 2011), SNP microarray (Hussein et al., 2011; Laurent et al., 2011), or next generation sequencing (Gore et al., 2011) Detailed analysis by using Ji et al., 2012) suggests that finer abnormalities, such as copy number variation (CNV) and mutations, occur in iPS cells at a higher frequency than originally thought. However, the exact load of mutations induced by cellular reprogramming is highly controversial (Bai et al., 2013). Nevertheless, hiPS may accumulate genetic changes during cell culture.

Draper, J. S., Moore, H. D., Ruban, L. N., Gokhale, P. J., Andrews, P. W., 2004. Culture and characterization of human embryonic stem cells. Stem Cells Dev. 13, 325-36 Taapken, SM, Nisler, BS, Newton, MA, Sampsell-Barron, TL, Leonhard, KA, McIntire, EM, Montgomery, KD, 2011. Karotypic abnormalities in human induced pluripotent stem cells and embryonic stem cells. Nat Biotechnol. 29, 313-4 Laurent, LC, Ulitsky, I., Slavin, I., Tran, H., Schork, A., Morey, R., Lynch, C., Harness, JV, Lee, S., Barrero, MJ, Ku, S ., Martynova, M., Semechkin, R., Galat, V., Gottesfeld, J., Izpisua Belmonte, JC, Murry, C., Keirstead, HS, Park, HS, Schmidt, U., Laslett, AL, Muller , FJ, Nievergelt, CM, Shamir, R., Loring, JF, 2011.Dynamic changes in the copy number of pluripotency and cell proliferation genes in human ESCs and iPSCs during reprogramming and time in culture.Cell Stem Cell. 8, 106- 18 Narva, E., Autio, R., Rahkonen, N., Kong, L., Harrison, N., Kitsberg, D., Borghese, L., Itskovitz-Eldor, J., Rasool, O., Dvorak, P ., Hovatta, O., Otonkoski, T., Tuuri, T., Cui, W., Brustle, O., Baker, D., Maltby, E., Moore, HD, Benvenisty, N., Andrews, PW, Yli-Harja, O., Lahesmaa, R., 2010. High-resolution DNA analysis of human embryonic stem cell lines reveals culture-induced copy number changes and loss of heterozygosity. Nat Biotechnol. 28, 371-7 Lefort, N., Feyeux, M., Bas, C., Feraud, O., Bennaceur-Griscelli, A., Tachdjian, G., Peschanski, M., Perrier, AL, 2008. human embryonic stem cells reveal recurrent genomic instability at 20q11.21. Nat Biotechnol. 26, 1364-6 Spits, C., Mateizel, I., Geens, M., Mertzanidou, A., Staessen, C., Vandeskelde, Y., Van der Elst, J., Liebaers, I., Sermon, K., 2008. Recurrent chromosomal abnormalities in human embryonic stem cells. Nat Biotechnol. 26, 1361-3 Cheng, L., Hansen, NF, Zhao, L., Du, Y., Zou, C., Donovan, FX, Chou, BK, Zhou, G., Li, S., Dowey, SN, Ye, Z. , Chandrasekharappa, SC, Yang, H., Mullikin, JC, Liu, PP, 2012. Low incidence of DNA sequence variation in human induced pluripotent stem cells generated by nonintegrating plasmid expression.Cell Stem Cell. 10, 337-44 Funk, WD, Labat, I., Sampathkumar, J., Gourraud, PA, Oksenberg, JR, Rosler, E., Steiger, D., Sheibani, N., Caillier, S., Stache-Crain, B., Johnson , JA, Meisner, L., Lacher, MD, Chapman, KB, Park, MJ, Shin, KJ, Drmanac, R., West, MD, 2012.Evaluating the genomic and sequence integrity of human ES cell lines; comparison to normal genomes. Stem Cell Res. 8, 154-64 Gore, A., Li, Z., Fung, HL, Young, JE, Agarwal, S., Antosiewicz-Bourget, J., Canto, I., Giorgetti, A., Israel, MA, Kiskinis, E., Lee , JH, Loh, YH, Manos, PD, Montserrat, N., Panopoulos, AD, Ruiz, S., Wilbert, ML, Yu, J., Kirkness, EF, Izpisua Belmonte, JC, Rossi, DJ, Thomson, JA , Eggan, K., Daley, GQ, Goldstein, LS, Zhang, K., 2011. Somatic coding mutations in human induced pluripotent stem cells. Nature. 471, 63-7 Martins-Taylor, K., Xu, R. H., 2010. Determinants of pluripotency: from avian, rodents, to primates.J Cell Biochem. 109, 16-25 Pasi, CE, Dereli-Oz, A., Negrini, S., Friedli, M., Fragola, G., Lombardo, A., Van Houwe, G., Naldini, L., Casola, S., Testa, G ., Trono, D., Pelicci, PG, Halazonetis, TD, 2011. Genomic instability in induced stem cells. Cell Death Differ. 18, 745-53 Hussein, SM, Batada, NN, Vuoristo, S., Ching, RW, Autio, R., Narva, E., Ng, S., Sourour, M., Hamalainen, R., Olsson, C., Lundin, K ., Mikkola, M., Trokovic, R., Peitz, M., Brustle, O., Bazett-Jones, DP, Alitalo, K., Lahesmaa, R., Nagy, A., Otonkoski, T., 2011. Copy number variation and selection during reprogramming to pluripotency.Nature. 471, 58-62 Ji, J., Ng, SH, Sharma, V., Neculai, D., Hussein, S., Sam, M., Trinh, Q., Church, GM, McPherson, JD, Nagy, A., Batada, NN , 2012. Elevated coding mutation rate during the reprogramming of human somatic cells into induced pluripotent stem cells.Stem Cells.30, 435-40

  These genetic abnormalities are of great concern because any DNA mutation can be a step in the malignant transformation process. Furthermore, certain abnormalities are highly recurrent, suggesting a strong selective pressure mediated by enhanced cell survival, cell proliferation or differentiation interference. These functional modifications increase the susceptibility of PSCs to malignant transformation and may change their expected therapeutic properties.

  The DNA integrity of pluripotent stem cells is mainly evaluated by karyotype analysis. Other approaches, such as CGH arrays or SNP microarrays, were tested to overcome the apparent resolution limitations of classical karyotyping; however, mutations and PSCs that could actually cause carcinogenesis There is no consensus on the methods used to distinguish between DNA modifications or simple polymorphisms with little or no potential to affect biological behavior. DNA sequencing technologies and their resolution (entire genome map in single base resolution) are improving very rapidly, and their prices are rapidly decreasing, so sometime PSC routine analysis will become whole genome sequencing You can expect to be dependent on Thing. However, each of these methods has strong limitations. For example, classical karyotyping methods are time consuming, require training of cytogenetics, and cannot detect abnormalities of length less than 5 Mb. Microarray-based approaches require core facilities and bioinformatics specialists dedicated to analysis. Finally, high-throughput sequencing methods, such as NGS, are not yet optimized for this application, require a long time to process the data, and also require bioinformatics specialists.

  Thus, there is a strong need for a quick and inexpensive non-invasive (cell-free) method that can detect most recurrent anomalies in hPSC.

The present invention relates to non-invasive methods and kits for determining the quality of pluripotent stem cells.
The invention also relates to non-invasive methods and kits for assessing gene integrity of cultured pluripotent stem cells.

Figure 1: Schematic diagram of the workflow. Assumed use of genomic analysis of supernatants to qualify cultured hPSCs. Collect hPSC supernatant and extract total cfDNA. PCR is then performed. The results are then analyzed by bioinformatics to detect biomarkers in cfDNA. FIG. 2: Reversion of hPSC gene abnormalities collected in SEAdb. Color gradient: # test; bubble size: length of genomic region; Y: recurrence score; X: chromosome. Figure 3: For 21 chromosomes carrying the most genetic alterations> 1 Mb, 40 sets of sequences in Table 1 (Sonses: S1-S40) cover 93.5% of chromosomal abnormalities. FIG. 4: Detection and quantification of cfNA in hPSC supernatant samples. Human ALU repeat sequence amplification in two hPSC supernatants using DNA from human foreskin fibroblasts at five concentrations (330 pg, 110 pg, 13 pg, 3.3 pg and 0.33 pg) as controls Evaluated. qPCR experiments were performed on a Roche LC480. Fluorescence was acquired at each cycle and plotted against the number of cycles. The increase in the amount of fluorescence measured is proportional to the amount of PCR product produced during the reaction. The measured concentration of cfNA in hPSC is 330 pg to 110 pg. FIG. 5: Detection of supernatant-based trisomy 20. A. Representative abnormal karyotypes in the two hPSC series: HD291 (47, XY, +12) (left panel) and HD129 (47, XY, +20) (right panel). B. DdPCR quantification of trisomy 20 in two hPSC series and their supernatants using a hyper-recurrent sequence specific for trisomy 20 only. Copy number plots using strict triple wells revealed the presence of trisomy 20 only in abnormal hPSC cells HD129 and their supernatants, but not in HD291. All error bars created by QuantaSoft ™ software represent 95% confidence intervals. FIG. 6: The sensitive QX200 system allows quantification of trisomy 20 in hPSC supernatants using specific hyperrecurrent sequences. The sample concentration is plotted as copy number / μl.

  Pluripotent stem cells (PSCs), which persist through self-renewal but can differentiate into mature cells of specific tissues, are a key tool for regenerative medicine. Regenerative medicine is a broad definition of innovative medicine that allows the body to repair, replace, restore and regenerate damaged or diseased cells, tissues and organs. However, cell culture can produce epigenetic and genetic abnormalities that can alter the properties of stem cells or cause them to predispose to tumorigenesis. As the clinical use of PSC expands rapidly, there is an opportunity to improve tools for characterizing pluripotent stem cells (PSCs) during cell growth and prior to batch release.

  We have determined a set of “hyperrecurrent sequences” that are biomarkers of hPSC instability in culture in human pluripotent stem cells (hPSCs) (Table 1), and the stem cells are in culture and prior to clinical use. Advocate a quick and easy-to-implement test that can be used to assess routines.

Table 1: List of 40 hyperrecurrent sequences Position represents the 5 'end of the amplification based on human genome build 37 (GRCh37 / hg19)

  Accordingly, the present invention relates to an in vitro non-invasive method comprising the following steps for determining the quality of pluripotent stem cells: i) providing a culture sample in which pluripotent stem cells are growing, ii) from the sample Nucleic acid is extracted, and iii) determining the presence and / or level of at least one genetic abnormality in the nucleic acid extract.

  As used herein, the term “pluripotent stem cell” or “PSC” has its general meaning and is a pluripotent cell that can differentiate into any cell of the human body, such as embryonic stem cells (ESC) and Represents induced pluripotent stem cells (iPSC). The term “pluripotency” refers to a cell that can differentiate into one of a wide variety of cell types, but not necessarily all cell types. Cells used in the present invention include, but are not limited to: cardiomyocytes and their progenitors; neural progenitors; pancreatic islets of Langerhans, in particular pancreatic β-cells; hematopoietic stem and progenitor cells; Stem cells; and muscle satellite cells. The method of the present invention can be applied to pluripotent stem cells, but can be applied to other stem cells, germ cells or somatic cells (eg, mesenchymal stem cells (MSC), oocytes, embryos, fibroblasts, etc.) Is also applicable.

  “Determining the quality of a pluripotent stem cell” means that the method of the present invention aims to determine whether a pluripotent stem cell has a genetic abnormality or a specific sequence related to regenerative medicine. Shall. The method of the present invention allows for the assessment of gene integrity and gene stability of cultured pluripotent stem cells.

  As used herein, the term “genetic abnormality” is any event that may be present in the genome of an individual and pluripotent stem cells, and refers to phenotypic disease and lethality. Represents what can be caused. Genetic abnormalities include, but are not limited to: trisomy, translocation, quadrisomy, aneuploidy, partial aneuploidy, monosomy, karyotypic abnormalities, isoforms Isodicentric chromosome, isochromosome, inversion, insertion, duplication, deletion, copy number variation (CNV), chromosome translocation, single nucleotide variation (SNV), And Loss of heterozygosity (LOH). In general, the term “gene abnormality” refers to the hyperrecurrent sequence described in Table 1, for example.

The term “culture sample” refers to the culture supernatant, medium, and cells suspended in the culture.
The term “nucleic acid” as used herein has its general meaning in the art and represents a coding or non-coding nucleic acid sequence. Nucleic acids include DNA (deoxyribonucleic acid) and RNA (ribonucleic acid). Thus, examples of nucleic acids include, but are not limited to, DNA, mRNA, tRNA, rRNA, tmRNA, miRNA, piRNA, snoRNA, and snRNA. The term “nucleic acid” also refers to free nucleic acid (fNA) (derived from the cell nucleus or cell mitochondrial compartment), such as cell-free DNA, free RNA molecules, microRNA, and long non-coding RNA. Involved. “Free nucleic acid” shall mean that the nucleic acid is released by the pluripotent stem cells and is present in the medium in which the pluripotent stem cells are growing.

To extract the released cellular nucleic acid from the prepared sample, one skilled in the art can use any method known in the art. For example, the methods described in the examples can be used.
In a particular embodiment, the method of the invention comprises the following steps: i) determining the presence of at least one hyperrecurrent sequence in the nucleic acid extract, and ii) if at least one hyperrecurrent sequence is detected, We conclude that pluripotent stem cells carry genetic abnormalities.

  Generally 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 hyperrecurrent sequences can be selected from Table 1.

  The determination of the presence and level of a hyperrecurrent sequence in a nucleic acid extract can be performed by various techniques well known in the art. In certain embodiments, a droplet digital-PCR “ddPCR” can be performed to determine the presence and level of hyperrecurrent sequences in a nucleic acid extract. ddPCR refers to the method or device used in that case that allows the quantification of DNA sequences in the supernatant or medium.

  The presence and level of the hyperrecurrent sequence in the nucleic acid extract can also be determined by techniques such as Fluidigm, quantitative PCR, high-throughput paired-end sequencing, next generation sequencing, and capillary electrophoresis.

  Representative methods for detecting hyperrecurrent sequences in nucleic acids, particularly DNA or mRNA, include, but are not limited to: restriction fragment length polymorphism, hybridization methods, sequencing, exonuclease Resistance, microsequencing, solid phase extension using ddNTPs, extension in solution using ddNTPs, oligonucleotide assays, methods for detecting single nucleotide polymorphisms such as dynamic allele-specific hybridization (dynamic allele- specific hybridisation), ligation chain reaction, mini-sequencing, DNA “chips”, allele-specific oligonucleotide hybridization with single- or double-labeled probes in combination with PCR or molecular beacons Other.

  In general, hyperrecurrent sequences are detected after amplification. For example, isolated RNA can be subjected to a combination of reverse transcription and amplification; polymerase chaining using specific oligonucleotide primers that are specific for or contain a hyperrecurrent sequence. Reverse transcription and amplification by reaction (RT-PCR). According to the first alternative, conditions can be chosen for specific reverse transcription (if appropriate) and primer annealing to ensure amplification; This is a diagnosis of the existence of a recurrent array. Alternatively, RNA can be reverse transcribed and amplified, or DNA can be amplified followed by hybridization with an appropriate probe, or directly sequencing or any other suitable method known in the art. Thus, the hyperrecurrent sequence in the amplified sequence can be detected. For example, cDNA obtained from RNA can be cloned and sequenced to identify hyperrecurrent sequences.

  In particular, sequencing is an ideal approach that can be used with the present invention. Those skilled in the art are familiar with several methods for sequencing polynucleotides. These include, but are not limited to: Sanger sequencing (also called dideoxy sequencing), and the various sequencing-by-synthesis (SBS) outlined by Metzger. Methods (Metzger ML 2005, Genome Research 1767), by hybridization, by ligation (eg, WO 2005/021786), by degradation (eg, US Patent Nos. 5,622,824 and 6,140,053), sequencing, nanopore sequencing. Preferably, deep sequencing is preferred in a multiplex assay. The term “deep sequencing” refers to a method of sequencing multiple nucleic acids in parallel. See, for example, Bentley et al, Nature 2008, 456: 53-59. By Roche / 454 (Margulies et al., 2005a), Illumina / Solexa (Bentley et al., 2008), Life / APG (SOLiD) (McKernan et al., 2009) and Pacific Biosciences (Eid et al., 2009) Major manufactured commercial platforms can be used for deep sequencing. For example, in the 454 method, DNA to be sequenced can be fractionated and supplied with an adapter, or a segment of DNA can be PCR amplified using a primer containing the adapter. The adapter is a nucleotide 25-mer required for binding to the DNA Capture Bead and for annealing the emulsion PCR amplification and sequencing primers. DNA fragments are made single stranded and attached to DNA capture beads so that one DNA fragment binds to one bead. The beads containing the DNA are then emulsified in a water-in-oil mixture, resulting in a microreactor containing only one bead. The fragments are PCR amplified in this microreactor, resulting in millions of copies per bead. After PCR, the emulsion is broken and the beads are loaded into a pico titer plate. Each well of the picotiter plate can contain only one bead. Sequencing enzyme is added to the wells and the nucleotides are flowed over the wells in a fixed order. As a result of nucleotide incorporation, pyrophosphate is released, which catalyzes the reaction that results in a chemiluminescent signal. This signal is recorded by a CCD camera, and the signal is converted into a DNA sequence using software. In the lllumina method (Bentley (2008)), a single-stranded adapter-supplied fragment is attached to an optically transparent surface and subjected to “bridge amplification”. This operation results in millions of clusters, each containing a copy of a unique DNA fragment. DNA polymerase, primers, and four types of labeled reversible terminator nucleotides are added and the surface is imaged by laser fluorescence to determine the location and nature of the label. The protecting group is then removed and the process is repeated for several cycles. The SOLiD method (Shendure (2005)) is similar to 454 sequencing and amplifies DNA fragments on the surface of the beads. Sequencing involves a cycle of labeled probe ligation and detection. Several other approaches for high-throughput sequencing are currently under development. Examples are Helicos system (Harris (2008)), Complete Genomics (Drmanac (2010)) and Pacific Biosciences (Lundquist (2008)). Since this is a very rapidly advancing technical field, the applicability of high-throughput sequencing methods to the present invention will be apparent to those skilled in the art.

  Determination of the expression level of nucleic acids (particularly genes, miRNA, snRNA, and snoRNA) can be evaluated by any of a variety of well-known methods. In general, the prepared nucleic acids can be used in hybridization or amplification assays, including but not limited to: Southern or Northern analysis, polymerase chain reaction analysis, such as quantitative PCR (TaqMan), and probe arrays, such as GeneChip probes. (Trademark) DNA array (AFF YMETRIX). Advantageously, the analysis of the expression level of the nucleic acid is carried out, for example, by RT-PCR (experimental embodiment described in US Pat. No. 4,683,202), ligase chain reaction (BARANY, Proc. Natl. Acad. Sci. USA, vol. 88). , p: 189-193, 1991), self sustained sequence replication (GUATELLI et al., Proc. Natl. Acad. Sci. USA, vol. 57, p: 1874-1878, 1990), transcription amplification System (KWOH et al., 1989, Proc. Natl. Acad. Sci. USA, vol. 86, p: 1173-1177, 1989), Q-beta replicase (LIZARDI et al., Biol. Technology, vol. 6, p: 1197, 1988), rolling circle replication (US Patent No. 5,854,033), or any other nucleic acid amplification process, followed by amplification molecules using techniques well known to those skilled in the art With detection. Real-time quantification or semi-quantitative RT-PCR is preferred. In certain embodiments, the determination includes hybridizing the sample with a selection reagent, such as a probe or primer, thereby detecting the presence or measuring the amount of the nucleic acid. Hybridization can be performed with any suitable device, such as a plate, microtiter dish, test tube, well, glass, column, and the like. Nucleic acids that show sequence complementarity or homology with the nucleic acid of interest in the present invention are useful as hybridization probes or amplification primers. Such nucleic acids need not be identical to homologous regions of equal size, but are generally interpreted as being at least about 80% identical, more preferably 85% identical, and even more preferably 90-95% identical. Is done. In certain embodiments, it may be advantageous to use the nucleic acid in combination with a suitable means for detecting hybridization, such as a detectable label. A variety of suitable indicators are known in the art, including fluorescent, radioactive, enzymatic or other ligands (eg, avidin / biotin). Probes and primers are “specific” for the nucleic acid to which they hybridize; that is, they are preferably under high stringency hybridization conditions (corresponding to the highest melting temperature—Tm—; for example, 50% formamide 5 × or 6 × SCC; 1 × SCC is 0.15M NaCl, 0.015M sodium citrate). A number of quantitative assays are commercially available from Qiagen (SA, Courteau, France) or Applied Biosystems (Foster City, USA). The expression level of the nucleic acid can be expressed as an absolute expression profile or a normalized expression profile. In general, the expression profile is normalized by correcting the absolute expression profile of the nucleic acid of interest by comparing its expression with the expression of an irrelevant nucleic acid, such as a constitutively expressed housekeeping mRNA. Suitable mRNAs for normalization include housekeeping mRNAs such as U6, U24, U48 and S18. This normalization allows for comparison of expression profiles in one sample, eg, a patient sample, with other samples, or between samples from different sources.

  Probes and / or primers are generally labeled with a detectable molecule or substance, such as a fluorescent molecule, a radioactive molecule, or any other label known in the art. Labels are generally known to generate signals (directly or indirectly). The term “labeled” refers to direct labeling of probes and primers by coupling (ie, physically linking) a detectable substance, and indirect by reactivity with other directly labeled reagents. It shall include labeling. Examples of detectable substances include, but are not limited to, radioactive materials or fluorophores (eg, fluorescein isothiocyanate (FITC) or phycoerythrin (PE) or indocyanine (Cy5)).

  The method of the present invention is particularly suitable for determining the quality of a pluripotent stem cell culture and then isolating pluripotent stem cells that do not contain a genetic abnormality. This method is particularly suitable for avoiding the degradation of pluripotent stem cell cultures containing pluripotent stem cells that do not contain genetic abnormalities as described above, which can be isolated and cultured.

Accordingly, the present invention relates to a method comprising the following steps for isolating pluripotent stem cells free of genetic abnormalities:
i) determining the level of hyperrecurrent sequence in a pluripotent stem cell culture by carrying out the method according to the invention,
ii) compare the level determined in step i) with a reference value;
iii) if the level determined in step i) is different from the reference value, conclude that the pluripotent stem cell culture contains pluripotent stem cells that do not contain a genetic abnormality,
iv) Isolating pluripotent stem cells that do not contain genetic abnormalities.

The step of isolating pluripotent stem cells can be performed by various techniques well known in the art, such as the fluidigm method.
In certain embodiments, the reference value is a threshold or cut-off value that can be determined experimentally, empirically, or theoretically. The threshold can be arbitrarily selected based on existing experimental conditions, as will be appreciated by those skilled in the art. In order to obtain optimal sensitivity and specificity, thresholds must be determined according to test function and benefit / risk balance (false positive and false negative clinical prognosis). In general, optimal sensitivity and specificity (and thus threshold) can be determined using a Receiver Operating Characteristic (ROC) curve based on experimental data. Preferably, one skilled in the art can compare the nucleic acid level (obtained according to the method of the invention) with the determined threshold. In one embodiment of the invention, the threshold is derived from the nucleic acid level (or ratio, or score) determined in a pluripotent stem cell culture carrying a genetic abnormality. In addition, retrospective measurements of nucleic acid levels (or ratios, or scores) in properly deposited historical pluripotent stem cell cultures can be used to establish these thresholds.

  The method of the present invention is particularly suitable for reaching clinical decisions. As used herein, the term “clinical decision” refers to the determination of whether to take an action with an outcome that affects the health or survival of a subject. In particular, in the context of the present invention, a clinical decision represents the determination of whether pluripotent stem cells will be transmitted, grafted, or transplanted to a subject. A clinical decision can also represent a decision to perform further testing to take action to reduce undesirable phenotypes. Thus, in particular, such methods would help clinicians avoid transmitting pluripotent stem cells carrying genetic abnormalities to a subject. The method described above is for avoiding contamination of a subject by pluripotent stem cells having a genetic abnormality, and for avoiding the onset of diseases such as malignant diseases caused by transmitting pluripotent stem cells having a genetic abnormality to a subject. Is also particularly appropriate. The method is also particularly suitable for treating a subject in need of administration without side effects by administering pluripotent stem cells.

  As used herein, the term “subject” refers to a mammal. In general, a subject according to the present invention represents any subject (preferably a human) in need of regenerative treatment using pluripotent stem cell transplantation. The term “subject” also refers to other mammals, such as primates, dogs, cats, pigs, cows, or mice. In certain embodiments, the term “subject” refers to a disease in need of regenerative treatment using pluripotent stem cell transplantation, such as spinal cord injury, Stargardt macular dystrophy (SMD), atrophic (dry) age-related macular degeneration, type I diabetes For subjects suffering from or susceptible to cardiovascular disorders such as heart failure, advanced Stargardt disease, wet (wet) age-related macular degeneration (AMD), muscular dystrophy, neurological and retinal diseases, liver disease, and diabetes Represent.

  Thus, the methods of the present invention allow an assessment of the ability of pluripotent stem cells to perform healthy transmission, grafting, and transplantation to a subject. The methods of the invention allow genetic testing and selection of pluripotent stem cells that can be transmitted, grafted, or transplanted into a subject.

  Pluripotent stem cells selected by carrying out the method of the present invention and differentiated cells derived therefrom can be used for regenerative medicine. The term “regenerative medicine” has its general meaning, a living functionality to repair or replace the function of cells, tissues or organs lost due to age, disease, injury, or birth defects Represents a regenerative procedure related to the process of creating cells and tissues.

  Accordingly, the present invention also relates to a method for transplanting pluripotent stem cells or differentiated cells derived therefrom to a subject in need of regenerative treatment comprising the following steps: i) performing the method according to the present invention, ii) selecting a pluripotent stem cell not containing a genetic abnormality, and iii) administering the pluripotent stem cell selected in step ii) or a differentiated cell derived therefrom to the subject.

  In a further aspect, the present invention is particularly suitable for treating disease using pluripotent stem cell transplantation in subjects in need of regenerative treatment, minimizing the risk of transmitting genetic abnormalities. The method of the present invention uses pluripotent stem cell transplantation in a subject in need of regenerative treatment, minimizing the risk of diseases such as malignant diseases caused by transplantation of pluripotent stem cells carrying a genetic abnormality. It is also suitable for treating diseases.

  Accordingly, the present invention also relates to a method for treating a disease in need of regenerative treatment in a subject comprising the following steps: i) performing the method according to the present invention, ii) pluripotency free from genetic abnormalities Stem cells are selected, and iii) pluripotent stem cells selected in step ii) or differentiated cells derived therefrom are administered to the subject.

  In a further aspect, the present invention relates to a method comprising the following steps for enhancing a response to regenerative treatment in a subject in need thereof: i) performing a method according to the present invention, ii) a gene free of genetic abnormalities A pluripotent stem cell is selected, and iii) the pluripotent stem cell selected in step ii) or a differentiated cell derived therefrom is administered to the subject.

  The invention also relates to a kit for carrying out the method, wherein the kit comprises means for determining the presence and / or level of at least one genetic abnormality in the nucleic acid extract. In general, the kit comprises the probe, primer, macroarray or microarray described above. For example, the kit can include a set of probes as defined above, which may be pre-labeled. Alternatively, the probe may not be labeled, and components for labeling may be accommodated in another container in the kit. The kit can further include hybridization reagents necessary for the hybridization protocol or other reagents and materials appropriately packaged (including a solid phase matrix if necessary), and standards. Alternatively, the kits of the invention can include amplification primers (eg, stem-loop primers), which can be pre-labeled, or can include an affinity purification or attachment moiety. The kit can further include amplification reagents necessary for the amplification protocol and other reagents and materials appropriately packaged. The kit can further include the means necessary to determine if amplification has occurred. The kit can also include, for example, PCR buffers and enzymes; positive control sequences, reaction control primers; and instructions for amplifying and detecting specific sequences.

  The invention is further illustrated by the following figures and examples. However, these examples and drawings should not be construed to limit the scope of the present invention in any way.

Example 1:
Method :
Culture and supernatant collection of hPSC Human PSC (hESC or iPSC) in a 35-mm well on Geltrex ™, xeno-free, completely defined medium (xeno-free) Cultured in the presence of (Essential 8 ™ medium). Cells were either mechanically dissociated and grown in bulk culture or enzymatically dissociated to suit single cell passage. The medium was changed every day. Medium without hPSC was incubated as a control. Immediately prior to PSC routine passage, 1 ml supernatant (hPSC conditioned medium) was collected from each well and immediately sterile, DNA-, DNase-, RNase-, polymerase chain reaction (PCR) inhibitor-free tubes And frozen at -80 ° C until nucleic acid purification. Appropriate care was taken to prevent sample contamination with foreign DNA.

Nucleic acid purification Nucleic acid was extracted from 200 μl of supernatant using QIAmp DNA Mini Blood Kit (Qiagen, Hilden, Germany) according to the preparation protocol. In summary, 20 μl of protease K and 200 μl of Buffer AL were added to each supernatant. After 15 seconds of pulse vortexing, the cell lysis mixture was incubated at 56 ° C. for 10 minutes in an Eppendorf tube (1.5 ml). High denaturation conditions at high temperatures were preferred for complete release of nucleic acids from any binding protein. After adding 200 μl of cold ethanol (100%) to the lysate, the sample was transferred onto a QIAamp Mini column. As the lysate was drawn by centrifugation at 6000 g for 1 minute, cell-free nucleic acid was adsorbed to the membrane. Contaminants were efficiently washed away during the two washing steps (in Buffer AW1 and Buffer AW2). Finally, cell-free nucleic acid was eluted in 30 μl Buffer AE and stored at −20 ° C.

Quantification of cell-free nucleic acid (cfNA) The concentration of cfNA in each supernatant was evaluated relative to the concentration of the corresponding ALU-115 PCR product determined by quantitative PCR (LC480, Roche). To this end, a total volume of 10 μL containing a commercially available 2 × LightCycler 480 SYBR Green I master mix (Roche Applied Science, Germany) and 0.25 μM forward and reverse ALU-primers described in Umetani et al. (2006). To each reaction mixture, 1 μl of each cfNA elution sample was added. Reactions were set up in white 96-well plates (Eppendorf) with an EpMotion 5070 liquid handling workstation (Eppendorf). All reactions were performed in triplicate. A negative control (water without RNAse / DNAse) was included in each experiment. The cfNA concentration in the supernatant was determined using a standard curve obtained with serial dilutions of genomic nucleic acid extracted directly from hPSC.

CNV detection using the digital droplet PCR system (ddPCR) in cfNA A ddPCR assay was performed as previously described (Abyzov et al. 2002). In summary, the ddPCR workflow consists of reaction setup, droplet creation, thermal cycling and running on the droplet reader according to the Bio-Rad instructions (Bio-Rad QX200 system). ddPCR utilizes a fluorescently labeled internal hybridization probe (TaqMan probe) for the detection of CNV in cfNA. The reaction is usually set up with one primer pair targeting the region of interest (eg: CNV-ID1, dHsaCP2506319) and a second primer pair targeting any reference gene (eg: RPP30, dHsaCP2500350). These two primers (target and reference) are labeled with different phosphors (FAM and HEX). The cfNA charge from each supernatant was added to the TaqMan PCR reaction mixture. Such a reaction mixture contained ddPCR Supermix No dUTP (Bio-Rad, Ref: 1863023) and primers in a final volume of 20 μl. Each constructed ddPCR reaction mixture was then loaded into the sample wells of an 8-channel disposable droplet generator cartridge (Bio-Rad, Ref: 1864008). A volume of 60 μL of droplet generating oil (Bio-Rad, Ref: 1863005) was loaded into the oil well for each channel. The cartridge was placed in a droplet generator (Bio-Rad). The cartridge was removed from the droplet generator and the droplets collected in the droplet wells were then manually transferred to a 96-well PCR plate with a multichannel pipette. The plate was heat sealed with a foil seal and then placed on a typical thermal cycler and amplified to the end point (40-50 cycles). Using microfluidics technology, the reaction mix is divided into spherical droplets composed of an oil surface and an aqueous core containing the PCR reaction mix. Apply thermal cycling to the droplets. After amplification, the fluorescence of each droplet is read continuously with a droplet reader. A droplet containing a target region or reference of interest (positive droplet) generates fluorescence in the corresponding channel, whereas a droplet not containing a target (negative droplet) does not generate fluorescence. The count of positive and negative droplets for each target is related to the concentration of the target in the sample by Poisson distribution.

Result :
Pluripotent stem cells (PSCs) that persist through self-replication but can differentiate into mature cells of specific tissues are key tools for regenerative medicine. Regenerative medicine is a broad definition of innovative medicine that allows the body to repair, replace, restore and regenerate damaged or diseased cells, tissues and organs. However, cell culture can produce epigenetic and genetic abnormalities that can alter the properties of stem cells or cause them to predispose to tumorigenesis. As the clinical use of PSC expands rapidly, there is an opportunity to improve tools for characterizing pluripotent stem cells (PSCs) during cell growth and prior to batch release.

  Currently, there are no reliable commercial genetic and non-invasive methods for assessing the gene integrity of cultured pluripotent stem cells. The present invention relates to a method for assessing the genomic integrity of cultured hPSC comprising the step of detecting a genetic abnormality of DNA present in the supernatant collected when the cultured PSC is growing.

  We have determined a set of “hyperrecurrent sequences” in hPSC that are biomarkers of cultured hPSC instability (Table 1) and used to routinely evaluate stem cells in culture and prior to clinical use. Propose a quick and easy-to-implement test that can be done (Figure 1).

Recurrent gene changes that occur during hPSC cultures. We have made it possible to contribute to the visualization of all types of genomic abnormalities obtained by karyotyping, FISH, microarray analysis (SNP, aCGH) or NGS. The database “SEAdb” was developed. SEAdb can be accessed via the following link: seadb.org (login: seadb and pwd: SEAdb). We collected abnormalities for over 400,000 abnormalities and variants.

We have shown that most recurrent gene changes that occur during hPSC culture are> 1 Mb karyotypic abnormalities and copy number variation (CNV) (FIG. 2).
In contrast, smaller genetic abnormalities, such as mutations and indels, are hardly recurrent. There are 1171 genetic changes in SEAdb> 1 Mb. We have selected a recurrency score that helps identify the location on the genome that is most susceptible to genomic modifications induced by PSC culture. For example, for 21 chromosomes harboring the majority of> 1 Mb genetic alterations, we have 40 pairs of sequences (Sonses: S1-S40) in Table 1 spanning 93.5% of chromosomal abnormalities. (FIG. 3).

Cell culture supernatant as a source of DNA for detecting pathogenic sequences The main constraint for assessing the genomic integrity of cultured stem cells is that the culture sample needs to be destroyed in order to perform the test . We therefore propose that we can examine the genomic integrity of cell culture supernatants.

  In fact, cell culture supernatants contain cell-free DNA (cfDNA) in the form of short fragments (70-200 base pairs in length), which are double-stranded molecules having a lower molecular weight than genomic DNA, or long fragments up to 21 kb. contains. Although the mechanism of cfDNA release is largely unknown, it has been suggested that necrosis, apoptosis, phagocytosis or active release play a role (Choi et al., 2005; Gahan et al., 2008; Stroun et al., 2001).

  cfDNA is present in serum or plasma and is used in non-invasive tests to examine chromosomal abnormalities (Hui and Bianchi, 2013). It has been demonstrated that certain fetal aneuploids such as trisomy 13, 18 or 21 can be detected in cell-free fetal DNA from maternal serum samples (Dan et al., 2012; Fairbrother et al., 2013; Nicolaides et al., 2014). In addition, fetal cfDNA in maternal plasma can also be used to detect pathogenic copy number variation (CNV) using target region capture sequencing (Ge et al., 2013).

  Based on the finding that cfDNA is released into various body fluids (serum, plasma) and can be used to detect pathogenic CNV, we have detected pathogenic CNV and non-invasive analysis of hPSCs. It is proposed to use DNA present in the supernatant avoiding cell destruction as a source for carrying out.

  In order to assess the potential of a foreign DNA source in the stem cell supernatant, we use quantitative real-time PCR of ALU repeats (Umetani et al., 2006). Quantification by ALU-qPCR of total cfDNA in two supernatants from two hESCs (triple) clearly indicates that cfDNA is detected in all test samples and the measured cfDNA concentration is 330 pg to 110 pg (FIG. 4).

  These results demonstrate that the hPSC supernatant contains cell-free DNA (cfDNA); it probably originates from the release of genetic material from dead cells and floating live cells. Detection of cfDNA released in hPSC supernatants is an unexplored tool to facilitate the assessment of genetic abnormalities using sequence biomarkers.

  The term “medium” relates to a nutrient solution for culturing, growing or growing cells. The term “cell culture” refers to cells that are maintained, cultured or grown in an artificial in vitro environment.

  The term “CNV” refers to a change in genomic DNA that results in cells with abnormal or normal variations for some genes in the copy number of one or more sections of DNA. A CNV corresponds to a relatively large genomic region that is deleted (less than normal) or duplicated (more than normal) on a particular chromosome.

Example 2:
Method:
Karyotyping Human pluripotent stem cells were dissociated with TryPLE Select (Life Technologies) and grown for 3 days to reach the mid-exponential phase. The single cells were then incubated for 90 minutes with 1 / 10,000 KaryoMAX® Colecmid ™ (Life Technologies) for metaphase arrest, followed by hypotonic swelling with 0.075 M KCl solution at 37 ° C. for 20 minutes, The mixture was fixed three times in ice-cooled methanol / glacial acetic acid (3: 1, vol / vol). Twenty microliters of the nuclear suspension is dropped onto a glass slide, air dried at 18.4 ° C. and 60% humidity, rehydrated in water for 5 minutes, and then in EARLE orange or 10 × EBSS at 87 ° C. Denatured for 55 minutes. The slides were then rinsed in cold water and stained with 3% GIEMSA for 3 minutes, rinsed 5 times and allowed to air dry. Spectral microscopy and analysis were performed using a Metafer Slide Scanning Platform (MetaSystem).

Analysis and statistics of ddPCR data The number of droplets recording fluorescence for the target specific assay (dHsaCP2506319) was compared to the counts obtained for the reference specific assay (dHsaCP2500350). The final copy number was calculated by applying Poisson statistics using the manufacturer's QuantaSoft Software (Bio-Rad, CA, USA):
λ = ln (1-p)
Here, “λ” is the average number of copies per droplet, and “p” is the ratio of positive droplets to the total number of droplets.

result:
Assessment of gene integrity in hPSC supernatants using ddPCR method: applied to routine screening Assessment of gene integrity screening is possible by examining cfDNA in hPSC supernatants. We confirmed the feasibility of this study using two aneuploid human pluripotent stem cell (hPSC) lines HD129 and HD291. As determined by general R-band karyotype determination, HD129 showed lysomy 20 (47, XY, +20), while HD291 showed trisomy 12 (47, XY, +12). Corresponding cells and supernatants were collected for our trisomy 20 analysis and ddPCR method using our specific hyperrecurrent sequence, respectively. As shown in FIG. 5, (i) genomic abnormality (trisomy 20 in this case) was detected in the hPSC supernatant using the ddPCR method for the HD129 hPSC series, but not detected in the HD291 series. A karyotype result is confirmed, (ii) a correlation is found between the genetic abnormality screening result from the supernatant and the corresponding karyotype, to evaluate the gene integrity of pluripotent stem cells Evidence of the concept that cfNA present in the supernatant can be used. An advantage of stem cell screening by using the supernatant would be that the integrity of the stem cell gene could be assessed without disruption. Furthermore, the use of this simple method based on the droplet digital polymerase chain reaction (ddPCR) makes it possible to screen the hPSC lineage quickly, efficiently and easily from a small amount of material including the culture supernatant. These benefits can make this method more attractive, providing the possibility of routine use. Finally, our method can be applied to any other experiment where rigorous genomic analysis is required for the examination of gene integrity (eg: pluripotency including mesenchymal stem cells (MSC) etc.) Sex stem cells, germ cells, lymphocytes, embryos, or somatic cells).

Nucleic acid minimum concentrations for reliable testing Nucleic acids at various concentrations extracted from supernatants collected from hPSC series HD129 (1, 1 ng / μL, 0, 4 ng / μL, 0, 1 ng / μL, 3, 7 pg / μL , 1,1 pg / μL, 0,4 pg / μL) was evaluated to evaluate the sensitivity of trisomy 20 sequence detection using ddPCR. As shown in FIG. 6, a trisomy 20 signal was detected between signals obtained from concentrations that were very low (as low as 0.1 ng / μL) but still sufficient to obtain reliable screening results. .

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Nucleic acid minimum concentrations for reliable testing Nucleic acids at various concentrations extracted from supernatants collected from hPSC series HD129 (1, 1 ng / μL, 0, 4 ng / μL, 0, 1 ng / μL, 3, 7 pg / μL , 1,1 pg / μL, 0,4 pg / μL) was evaluated to evaluate the sensitivity of trisomy 20 sequence detection using ddPCR. As shown in FIG. 6, a trisomy 20 signal was detected between signals obtained from concentrations that were very low (as low as 0.1 ng / μL) but still sufficient to obtain reliable screening results. .
In certain embodiments, the present invention may be:
[Aspect 1] In vitro non-invasive method including the following steps for determining the quality of pluripotent stem cells: i) preparing a culture sample in which pluripotent stem cells are proliferating; ii) extracting nucleic acid from the sample And iii) determining the presence and / or level of at least one genetic abnormality in the nucleic acid extract.
[Aspect 2] i) Determine the presence of at least one hyperrecurrent sequence selected from Table 1 in the nucleic acid extract, and ii) if at least one hyperrecurrent sequence is detected, the pluripotent stem cell is a gene The method of embodiment 1, comprising the step of concluding that the disorder is retained.
[Aspect 3] A method comprising the following steps for isolating pluripotent stem cells not containing a genetic abnormality:
i) determining the level of hyperrecurrent sequence in the pluripotent stem cell culture by performing the method of aspect 2;
ii) compare the level determined in step i) with a reference value;
iii) if the level determined in step i) is different from the reference value, conclude that the pluripotent stem cell culture contains pluripotent stem cells that do not contain a genetic abnormality, and iv) a pluripotent that does not contain a genetic abnormality Isolate sex stem cells.
[Aspect 4] A method comprising the following steps for transplanting pluripotent stem cells or differentiated cells derived therefrom to a subject in need of regenerative treatment: i) The method according to any one of aspects 1 to 3 Ii) selecting pluripotent stem cells that do not contain a genetic abnormality, and iii) administering the pluripotent stem cells selected in step ii) or differentiated cells derived therefrom to the subject.
[Aspect 5] A method comprising the following steps for treating a disease in need of regenerative treatment in a subject: i) performing the method according to any one of aspects 1 to 3, ii) including a genetic abnormality A pluripotent stem cell is selected, and iii) the pluripotent stem cell selected in step ii) or a differentiated cell derived therefrom is administered to the subject.

Claims (5)

  1.   In vitro non-invasive method comprising the following steps for determining the quality of pluripotent stem cells: i) providing a culture sample in which pluripotent stem cells are growing, ii) extracting nucleic acid from the sample, and iii ) Determine the presence and / or level of at least one genetic abnormality in the nucleic acid extract.
  2.   i) determining the presence of at least one hyperrecurrent sequence selected from Table 1 in the nucleic acid extract, and ii) if at least one hyperrecurrent sequence is detected, the pluripotent stem cell carries a genetic abnormality The method of claim 1, comprising the step of concluding.
  3. A method comprising the following steps for isolating pluripotent stem cells free of genetic abnormalities:
    i) determining the level of hyperrecurrent sequence in a pluripotent stem cell culture by performing the method of claim 2;
    ii) compare the level determined in step i) with a reference value;
    iii) if the level determined in step i) is different from the reference value, conclude that the pluripotent stem cell culture contains pluripotent stem cells that do not contain a genetic abnormality, and iv) a pluripotent that does not contain a genetic abnormality Isolate sex stem cells.
  4.   A method comprising the following steps for transplanting pluripotent stem cells or differentiated cells derived therefrom to a subject in need of regenerative treatment: i) performing the method according to any one of claims 1 to 3 Ii) selecting a pluripotent stem cell not containing a genetic abnormality, and iii) administering the pluripotent stem cell selected in step ii) or a differentiated cell derived therefrom to the subject.
  5. A method comprising the following steps for treating a disease in need of regenerative treatment in a subject: i) performing the method according to any one of claims 1 to 3, ii) having no gene abnormality A pluripotent stem cell is selected, and iii) the pluripotent stem cell selected in step ii) or a differentiated cell derived therefrom is administered to the subject.
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